Tai-Tung Yip

Mapping and sequence-specific identification of phosphopeptides in unfractionated protein digest mixtures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

We have demonstrated a procedure for the rapid (minutes), sensitive (

Occupancy of a C2-C2 type ‘Zinc-finger’ protein domain by copper Direct observation by electrospray ionization mass spectrometry☆

The metal ion specificity of most ‘zinc‐finger’ metal binding domains is unknown. The human estrogen receptor protein contains two different C2‐C2 type ‘zinc‐finger’ sequences within its DNA‐binding domain (ERDBD). Copper inhibits the function of this protein by mechanisms which remain unclear. We have used electrospray ionization mass spectrometry to evaluate directly the 71‐residue ERDBD (K180‐M250) in the absence and presence of Cu(II) ions. The ERDBD showed a high affinity for Cu and was completely occupied with 4 Cu bound; each Cu ion was evidently bound to only two ligand residues (net loss of only 2 Da per bound Cu). The Cu binding stoichiometry was confirmed by atomic absorption. These results (i) provide the first direct physical evidence for the ability of the estrogen receptor DNA‐binding domain to bind Cu and (ii) document a twofold difference in the Zn‐ and Cu‐binding capacity. Differences in the ERDBD domain structure with bound Zn and Cu are predicted. Given the relative intracellular contents of Zn and Cu, our findings demonstrate the need to investigate further the Cu occupancy of this and other zinc‐finger domains both in vitro and in vivo.

Rapid purification of porcine colostral whey lactoferrin by affinity chromatography on single-stranded DNA-agarose. Characterization, amino acid composition and N-terminal amino acid sequence

We have determined that the major iron-binding and DNA-binding protein in porcine colostral whey is lactoferrin. This lactoferrin was purified to homogeneity in one chromatographic step using immobilized single-stranded DNA-agarose. Although different in chromatographic behavior from human lactoferrin, the porcine lactoferrin purified in this manner was shown to be homogeneous by high-performance ion-exchange chromatography (Mono-S), immobilized metal ion (Cu2+) affinity chromatography, size-exclusion chromatography (TSK-4000SW), and reverse-phase (phenyl) chromatography. Electrophoresis on SDS-polyacrylamide gradient (10-20%) gels under reducing conditions showed the purified lactoferrin to be a single protein (silver-stained) of 78 kDa. Apolactoferrin purified in this manner bound iron and displayed a UV/VIS absorption spectrum indistinguishable from that of human lactoferrin. The molar absorption coefficient of hololactoferrin was 3.86 x 10(3) M-1 at 465 nm and 1.08 x 10(5) M-1 at 280 nm. Affinity elution analyses of the purified lactoferrin on immobilized DNA revealed that the affinity of this protein for DNA was independent of bound iron. Porcine lactoferrin was recognized by antibodies directed against human lactoferrin and bovine lactoferrin. The amino acid composition and N-terminal amino acid sequence analysis (30 residues) revealed a high degree of sequence homology with human, equine and bovine lactoferrin. These results demonstrate the effectiveness of immobilized DNA as a rapid and simple lactoferrin purification procedure and demonstrate the presence of a lactoferrin in porcine colostral whey with a high degree of sequence homology to human lactoferrin.

Metal ligand-induced alterations in the surface structures of lactoferrin and transferrin probed by interaction with immobilized copper(II) ions.

We have evaluated immobilized Cu(II) ions as a potential site-directed molecular probe to monitor ligand-induced alterations in protein surface structures. Metal ion-induced alterations in the surface structures of different lactoferrins (human and porcine), transferrins (human and rabbit), and ovotransferrin (chicken) were examined. Although these 78,000-dalton glycoproteins are related gene products with similar overall structure and function, they differ greatly in the number and distribution of surface-exposed electron-donor groups thought to interact with Cu(II) ions. Each of these proteins interacted with immobilized Cu(II) ions through sites which are distinct from the two specific high affinity metal binding sites identified for iron. In both the presence and absence of bound iron, transferrins interacted more strongly with the immobilized Cu(II) ions than did lactoferrins; ovotransferrin interacted only weakly. Although iron binding increased the affinities of lactoferrins for immobilized Cu(II), iron binding decreased the affinities of transferrins and ovotransferrin for immobilized Cu(II) ions. Iron-saturated and iron-free lactoferrins were resolved by pH gradient elution, but only in the presence of 3 M urea; they were not resolved by imidazole affinity elution. Conversely, the iron-saturated and iron-free forms of transferrin were only separated by imidazole affinity elution. Urea did not influence the resolution of apo and holo ovotransferrins by imidazole. The differential effects of urea and imidazole suggest the participation of different types of surface electron-donor groups. The progressive site-specific modification of surface-exposed histidyl residues by carboxyethylation revealed several lactoferrin forms of intermediate affinity for immobilized iminodiacetate-Cu(II) ions. In summary, independent of species, the affinity for immobilized Cu(II) ions increased as follows: iron-saturated ovotransferrin less than metal-free ovotransferrin less than apolactoferrin less than hololactoferrin much less than diferric or holotransferrin less than monoferric transferrin less than apotransferrin. We have demonstrated the use of immobilized Cu(II) ions to distinguish and to monitor ligand-induced alterations in protein surface structure. The results are discussed in relation to protein surface-exposed areas of electron-donor groups.

Metal ion affinity adsorption of a Zn(II)-transport protein present in maternal plasma during lactation: structural characterization and identification as histidine-rich glycoprotein.

A high-affinity Zn(II)-binding protein has been purified to homogeneity (880-fold) from the plasma of lactating women by a single affinity adsorption step on columns of tris(carboxymethyl)ethylenediamine (TED)-agarose loaded with Zn(II) ions. Purity was evaluated by high-performance reverse-phase (phenyl) chromatography and by silver staining after SDS-polyacrylamide gradient gel electrophoresis. The mass of denatured Zn(II)-binding protein was estimated by SDS-polyacrylamide gradient gel electrophoresis to be 75 kDa under both reducing and nonreducing conditions; by matrix-assisted uv laser desorption time-of-flight mass spectrometry the purified protein mass was determined to be 66 kDa. The amino acid composition revealed a high content of His (13 mol%) and Pro (12 mol%). N-terminal amino acid sequence analysis (50 residues) identified the purified protein as histidine-rich glycoprotein (HRG). Immunoblots demonstrated the absence of fragments in the purified product. An enzyme-linked immunosorbent assay was developed; a 75% recovery of intact HRG from the immobilized Zn(II) ion affinity column was documented. The circular dichroism spectra for the purified human HRG in the far uv (260-178 nm) were similar to those published for human and rabbit serum HRG. These results demonstrate that TED-immobilized Zn(II) ions can be used as a new and efficient method for the isolation of structurally intact human plasma HRG.

Synthetic Protein Surface Domains as Bioactive Stationary Phases

Significant developments toward the site- or domain-specific interaction of proteins with chemically defined stationary phases have been made during the last 10 to 20 years. Most of these efforts, however, were focused primarily on the need for improved protein purification strategies. One example was the introduction of new chemical methods for the immobilization of transition-metal ions (Porath et al., 1975; Porath and Olin, 1983). Immobilized metal ion affinity chromatography (IMAC), although relatively slow to gain widespread acceptance, is clearly an important advancement in the field of protein purification. In our view, however, the use of immobilized metal ions can address questions that go beyond affinity chromatography and protein purification. Indeed, the interactions of peptides and proteins with surface-immobilized transition-metal ions presents an opportunity to investigate and ultimately model biospecific metal ion-dependent macromolecular recognition events. To help explore the development of this opportunity, we have evaluated (1) protein surface—metal ion interaction mechanisms (Hutchens et al., 1988, 1989b; Hutchens and Li, 1988; Hutchens and Yip, 1990a,b, 1992a,b; Yip et al., 1989; Yip and Hutchens, 1989), (2) metal ion-dependent macromolecular surface recognition (Hutchens et al., 1981, 1989b), and (3) the use of immobilized metal ions to evaluate ligand-induced alterations in macro-molecular surface structure (Hutchens and Li, 1990; Hutchens and Yip, 1991b). We have shown both qualitative and quantitative examples of variability in affinity and selectivity in the biomolecular surface recognition of surface-immobilized metal ions. Until recently, our efforts in this area have been limited to the use of relatively simple, chemically defined (i. e., nonbiological) stationary-phase metal ion chelators of the type introduced by Porath and co-workers (Porath et al., 1975; Porath and Olin, 1983).